With regards to the first aspect of the invention, the promotion of selected cellular functions is an important task in a variety of applications, such as the production of undifferentiated mammalian embryonic stem cells and/or their uniform differentiated growth. In particular, the therapeutic uses of mammalian embryonic stem [ES] cells has attracted considerable attention, and there has evolved an increasing need for producing undifferentiated mammalian ES cells as well as methods to guide and control their differentiation. Consequently, suitable microenvironments facilitating/promoting these processes are desirable. Biocompatible materials, on which ES cells can attach, grow, and/or differentiate and/or further perform diverse biological functions, are thus needed for a variety of therapeutic purposes. Medical conditions whose treatment might benefit from such materials include degenerative disorders, cancer and trauma of the musculoskeletal apparatus, each of which constitutes an increasing problem in public health.
There are three major demands on ES cell culturing protocols. Firstly, during ES cell culturing, the cells have to receive the proper stimuli from soluble factors in the medium and from the growth support to maintain pluripotency. Secondly, chromosomal integrity should be maintained. Thirdly, in order to facilitate the use of mammalian ES cells for medical purposes it is essential that the cells at no point come into contact with biological materials derived from another species, since xeno-contamination is most likely to cause immunogenic problems upon transplantation into a patient. Current culturing protocols generally depend on the use of biological material of animal origin, where ES cells are grown on a layer of feeder cells and serum. Feeder- and serum-free culture conditions have been described for murine ES cells, for example using gelatin-coated dishes combined with Leukemia Inhibitory Factor (LIF)-supplemented media, but these culture systems are expensive and do not always give rise to ES cells suitable for all forms of therapy.
Furthermore, future medical treatments aim to employ differentiated ES cells for implantation into a patient. For this purpose it is essential to ensure uniform differentiation in all ES cells, since the presence of undifferentiated cells in the implant can give rise to teratomas in the patient, which remains a problem for current differentiation protocols.
In conclusion there is a great need to develop xeno-free ES cell culturing conditions, both conditions in which the cells maintain pluripotency and chromosomal integrity and conditions in which the cells differentiate in a uniform and controlled manner.
With regards to the second aspect of the invention, the promotion of selected cellular functions is an important task in a variety of applications, such as the development of suitable implants. Biocompatible materials, on which living cells can attach, grow, and/or differentiate and/or further perform diverse biological functions, are desirable for a variety of therapeutic purposes.
Degenerative disorders, cancer and trauma of the musculoskeletal apparatus constitute an increasing problem in public health. Spinal disorders alone affect 30 percent of the adult population, and 40 percent of those older than 65 years have symptoms of osteoarthritis. More than 1.3 million joint alloplasties are performed annually worldwide to treat debilitating end-stage arthritis. Since there are no accepted therapies to prevent osteoarthritis, it is anticipated that the number of arthroplasties performed will rise dramatically over the next several decades, due to the aging of the western population. At the present time, more than 25 percent of all health care expenditures in Europe and USA are related to musculoskeletal conditions, and the budgets to treat such disorders in the USA (254 Billion USD) are for instance double the resources used for research and teaching in total.
The main surgical treatments of these disorders rely on the use of metallic medical implants in conjunction with bone or bone substitutes. The implants must be successfully incorporated in the bone tissue in order to obtain good clinical results. Major advances and results have been achieved in this area during the last decades, but implant loosening over time continues to be a significant problem for successful long-term joint replacements. The current implant surfaces, alone, are not able to bridge larger bone defects and maintain long-term stability. The use of bone grafts taken from the patients themselves to solve these problems is followed by a high donor site morbidity of 15-30 percent. As many as 20% of the patients undergoing hip replacement develop bone loss around the prosthesis within 10 to 15 years of the initial surgery, and in spinal fusion surgery 20-30 percent of the patients obtain poor fusion. Furthermore, as the near-future patient population will include a significant number of younger patients, the problem concerning long-term aseptic implant loosening is predicted to increase dramatically.
Improvement of implant behavior in bone tissue will therefore have a tremendous impact, both in terms of quality of life and economy. The WHO has recognized this by appointing the years 2000-2010 as the “Bone and Joint Decade” (bonejointdecade.org/), an initiative also approved by the Danish Ministry of Health.
The biocompatibility/biointegration of an implant in the body is extremely complicated, involving processes traditionally belonging to medical science, surface science, materials science, and molecular biotechnology. When an implant is placed in tissue, a race for the surface starts immediately. Within a few milliseconds after the implant is inserted into the body, a biolayer consisting of water, proteins and other biomolecules from the physiological liquid is formed on the implant surface. Subsequently, cells from the surrounding tissue migrate to the area around the implant due to stimulation by cytokines and growth factors in the biolayer. The interaction between an implant surface and the cells is thus mediated through this biolayer. The properties of the implant surface strongly influence the properties of the layer and this influence needs to be understood and controlled in order to optimize biocompatibility. Of equal importance are the properties of the cells, e.g. their ability to communicate through the extracellular matrix by signal molecules. During bone healing, numerous bioactive signal molecules control bone formation and some proteins are found capable of stimulating bone healing to implants. All these mechanisms contribute to the response of the tissue to the implant and influence whether the implant is successfully anchored with sufficient mechanical strength in the bone of the patient or whether an inflammatory reaction against the implant occurs, which finally results in aseptic loosening and operative failure.
Biocompatible materials, on which bone tissue cells, can attach, and/or grow, and further perform diverse biological functions, are required for therapeutic purposes, in particular in surgical treatments involving the introduction of implants, such as prostheses and bone substitutes. Achieving a successful outcome of such treatment presents a formidable challenge, since an implant needs to allow tissue regeneration at the implant site, while avoiding becoming a target for the body's own powerful rejection mechanisms. The clinical success of an implant depends of the cellular behavior in the immediate vicinity of the interface between an implant and the host tissue. A key element in the progress in this field thus relies on the identification and use of a biocompatible material in the fabrication of these implants.
Bone tissue comprises a number of cell types including osteoprogenitor cells. Marrow stromal cells (MSCs) are pluripotent stem cells that give rise to both osteoprogenitor cells and other cell types. Osteoprogenitor cells can differentiate and form osteoblasts, particularly in response to bone regeneration. Bone modeling proteins (BMP and other growth hormones), produced by the marrow stromal cells, serve to both recruit osteoprogenitor cells and stimulate their maturation into osteoblasts. Osteoblasts secrete e.g. TGF-beta BMP's, other hormones and growth factors etc., which act both as a chemotactic attractant for osteoprogenitor cells, and stimulate the maturation of osteoblasts and induce the formation of bone matrix. Osteoblasts synthesize and secrete organic bone matrix (like collagen fibers, proteoglycans, osteocalcin, osteonectin and osteopontin) and hence osteoblasts play a key role in the deposition of mineralized bone matrix.
During the mineralization of bone, osteoblasts express alkaline phosphatase, together with a number of cytokines and growth hormones.
In the ongoing development of materials with improved biocompatibility there remains a need to identify materials whose structure is compatible with implant surgery and inductive for bone regeneration.
Furthermore, during recent years, therapeutic uses of embryonic stem cells has attracted considerable attention, and there has evolved an increasing need for the guided, controlled differentiation of embryonic stem cells. Consequently, suitable microenvironments facilitating/promoting these processes are desirable.